Bone Morphogenetic Protein 10-A Novel Biomarker to Predict Adverse Outcomes in Patients With Atrial Fibrillation.

Background Patients with atrial fibrillation (AF) face an increased risk of death and major adverse cardiovascular events (MACE). We aimed to assess the predictive value of the novel atrial-specific biomarker BMP10 (bone morphogenetic protein 10) for death and MACE in patients with AF in comparison with NT-proBNP (N-terminal prohormone of B-type natriuretic peptide). Methods and Results BMP10 and NT-proBNP were measured in patients with AF enrolled in Swiss-AF (Swiss Atrial Fibrillation Study), a prospective multicenter cohort study. A total of 2219 patients were included (median follow-up 4.3 years [interquartile range 3.9, 5.1], mean age 73±9 years, 73% male). In multivariable Cox proportional hazard models, the adjusted hazard ratio (aHR) associated with 1 ng/mL increase of BMP10 was 1.60 (95% CI, 1.37-1.87) for all-cause death, and 1.54 (95% CI, 1.35-1.76) for MACE. For all-cause death, the concordance index was 0.783 (95% CI, 0.763-0.809) for BMP10, 0.784 (95% CI, 0.765-0.810) for NT-proBNP, and 0.789 (95% CI, 0.771-0.815) for both biomarkers combined. For MACE, the concordance index was 0.732 (95% CI, 0.715-0.754) for BMP10, 0.747 (95% CI, 0.731-0.768) for NT-proBNP, and 0.750 (95% CI, 0.734-0.771) for both biomarkers combined. When grouping patients according to NT-proBNP categories (<300, 300-900, >900 ng/L), higher aHRs were observed in patients with high BMP10 in the categories of low NT-proBNP (all-cause death aHR, 2.28 [95% CI, 1.15-4.52], MACE aHR, 1.88 [95% CI, 1.07-3.28]) and high NT-proBNP (all-cause death aHR, 1.61 [95% CI, 1.14-2.26], MACE aHR, 1.38 [95% CI, 1.07-1.80]). Conclusions BMP10 strongly predicted all-cause death and MACE in patients with AF. BMP10 provided additional prognostic information in low- and high-risk patients according to NT-proBNP stratification. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02105844.

Sex disparities in patients with suspected COVID-19 presenting at an emergency department in Switzerland.

AIMS OF THE STUDY: In the global COVID-19 pandemic, female sex is associated with comparable infection rates but better outcome. However, most studies lacked appropriate controls. We investigated whether these sex disparity findings are specific to patients with COVID-19 or generalizable to patients presenting to the emergency room (ER) with similar symptoms but no COVID-19. METHODS: In this prospective cohort study, consecutive patients presenting with symptoms suggestive of COVID-19 were recruited at the ER of the University Hospital Basel, Switzerland from March to June 2020. Patients were categorized as SARS-CoV-2 positive (cases) or negative (controls) based on nasopharyngeal PCR swab tests. The final clinical diagnosis was determined for all patients. The primary outcome was a composite of intensive care admission, rehospitalization for respiratory distress and all-cause death within 30 days. We used Kaplan-Meier curves and Cox proportional hazards models to explore associations between sex and outcomes. RESULTS: Among 1,081 consecutive ER patients, 191 (18%) tested positive for SARS-CoV-2, with an even sex distribution (17.9% female vs. 17.5% male, p = 0.855). In COVID-19 patients, female sex was associated with lower risk of hospitalization (51% vs. 66%, p = 0.034), lower necessity of haemodynamic support (8% vs. 20%, p = 0.029), lower rates of intubation (10% vs. 21%, p = 0.037) and the primary outcome (18% vs. 31%, p = 0.045; age-adjusted HR 0.536, 95%CI 0.290-0.989, p = 0.046) compared with male sex. Sex disparities were most prominent in patients ≥55 years (HR for composite primary outcome in women 0.415, 95%CI 0.201-0.855, p = 0.017). In contrast to the COVID-19 patients, no sex-specific differences in outcomes were observed in the unselected overall control group (age-adjusted HR 0.844, 95%CI 0.560-1.273, p = 0.419) or in a subgroup of controls with upper respiratory tract infections or pneumonia (age-adjusted HR 0.840, 95%CI 0.418-1.688, p = 0.624). CONCLUSION: In this unselected, consecutive cohort study at a tertiary hospital in Switzerland, female sex is associated with better outcome in patients presenting to the ER with COVID-19. These sex disparities seem to be at least partly specific to COVID-19, as they were not observed in comparable controls.

Sex-specific differences in adverse outcome events among patients with atrial fibrillation.

OBJECTIVE: To assess whether women with atrial fibrillation (AF) have a higher risk of adverse events than men during long-term follow-up since controversial data have been published. METHODS: In the context of two very similar observational multicentre cohort studies, we prospectively followed 3894 patients (28% women) with previously documented AF for a median of 4.02 (3.00-5.83) years. The primary outcome was a composite of ischaemic stroke, myocardial infarction and cardiovascular death. Secondary outcomes included the individual components of the composite outcome, hospitalisation for heart failure, major and clinically relevant non-major bleeding, stroke or systemic embolism and non-cardiovascular death. RESULTS: Mean age was 73.1 years in women vs 70.8 years in men. The incidence of the primary endpoint in women versus men was 2.46 vs 3.24 per 100 patient-years, respectively (adjusted HR (aHR) 0.74, 95% CI 0.58 to 0.94; p=0.01). Women died less frequently from cardiovascular (aHR 0.57, 95% CI 0.41 to 0.78; p<0.001) and non-cardiovascular causes (aHR 0.68, 95% CI 0.47 to 0.98; p=0.04). There were no significant sex-specific differences in stroke (incidence 1.05 vs 1.00; aHR 1.02, 95% CI 0.70 to 1.49, p=0.93), myocardial infarction (incidence 0.67 vs 0.72; aHR 0.98, 95% CI 0.61 to 1.57, p=0.94), major and clinically relevant non-major bleeding (incidence 4.51 vs 4.34; aHR 0.95, 95% CI 0.79 to 1.15, p=0.63) or heart failure hospitalisation (incidence 3.28 vs 3.07; aHR 1.06, 95% CI 0.85 to 1.32, p=0.60). CONCLUSION: In this large study of patients with established AF, women had a lower risk of death than men, but there were no sex-specific differences in other adverse outcomes.

Silent brain infarcts impact on cognitive function in atrial fibrillation.

AIMS: We aimed to investigate the association of clinically overt and silent brain lesions with cognitive function in atrial fibrillation (AF) patients. METHODS AND RESULTS: We enrolled 1227 AF patients in a prospective, multicentre cohort study (Swiss-AF). Patients underwent standardized brain magnetic resonance imaging (MRI) at baseline and after 2 years. We quantified new small non-cortical infarcts (SNCIs) and large non-cortical or cortical infarcts (LNCCIs), white matter lesions (WML), and microbleeds (Mb). Clinically, silent infarcts were defined as new SNCI/LNCCI on follow-up MRI in patients without a clinical stroke or transient ischaemic attack (TIA) during follow-up. Cognition was assessed using validated tests. The mean age was 71 years, 26.1% were females, and 89.9% were anticoagulated. Twenty-eight patients (2.3%) experienced a stroke/TIA during 2 years of follow-up. Of the 68 (5.5%) patients with ≥1 SNCI/LNCCI, 60 (88.2%) were anticoagulated at baseline and 58 (85.3%) had a silent infarct. Patients with brain infarcts had a larger decline in cognition [median (interquartile range)] changes in Cognitive Construct score [-0.12 (-0.22; -0.07)] than patients without new brain infarcts [0.07 (-0.09; 0.25)]. New WML or Mb were not associated with cognitive decline. CONCLUSION: In a contemporary cohort of AF patients, 5.5% had a new brain infarct on MRI after 2 years. The majority of these infarcts was clinically silent and occurred in anticoagulated patients. Clinically, overt and silent brain infarcts had a similar impact on cognitive decline. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02105844, https://clinicaltrials.gov/ct2/show/NCT02105844.

Cardiac autonomic function and cognitive performance in patients with atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is associated with loss of cognition and dementia. Cardiac autonomic dysfunction has been linked to cognitive decline. We aimed to investigate if reduced cardiac autonomic function (CAF) is associated with cognitive impairment in AF patients. METHODS: Patients with paroxysmal, persistent and permanent AF were enrolled from a multicenter cohort study if they had AF ("AF group") or sinus rhythm ("SR group") on a baseline 5 min ECG recording. Parameters quantifying CAF (heart rate variability triangular index (HRVI), mean heart rate (MHR), RMSSD, SDNN, total power and power in the VLF, LF, HF ranges) were calculated. We used the Montreal Cognitive Assessment (MoCA) to assess global cognitive function. RESULTS: 1685 AF patients with a mean age of 73 ± 8 years, 29% females, were included. MoCA score was 24.5 ± 3.2 in the AF group (N = 710 patients) and 25.4 ± 3.2 in the SR group (N = 975 patients). After adjusting for multiple confounders, lower HRVI was associated with lower MoCA scores, both in the SR group [ß = 0.049; 95% confidence interval (CI) 0.016-0.081; p = 0.003] and in the AF group (ß = 0.068; 95% CI 0.020-0.116; p = 0.006). In the AF group, higher MHR was associated with a poorer performance in the MoCA (ß = - 0.008; 95% CI - 0.014 to - 0.002; p = 0.014). We found no convincing evidence of association for other CAF parameters with cognition. CONCLUSION: Our data suggest that impaired CAF is associated with worse cognitive performance in patients with AF. Among standard HRV parameters, HRVI might be the most promising ECG index. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02105844.

Direct Comparison of Clinical Characteristics, Outcomes, and Risk Prediction in Patients with COVID-19 and Controls-A Prospective Cohort Study.

Most studies investigating early risk predictors in coronavirus disease 19 (COVID-19) lacked comparison with controls. We aimed to assess and directly compare outcomes and risk predictors at time of emergency department (ED) presentation in COVID-19 and controls. Consecutive patients presenting to the ED with suspected COVID-19 were prospectively enrolled. COVID-19-patients were compared with (i) patients tested negative (overall controls) and (ii) patients tested negative, who had a respiratory infection (respiratory controls). Primary outcome was the composite of intensive care unit (ICU) admission and death at 30 days. Among 1081 consecutive cases, 191 (18%) were tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 890 (82%) were tested negative (overall controls), of which 323 (30%) had a respiratory infection (respiratory controls). Incidence of the composite outcome was significantly higher in COVID-19 (23%) as compared with the overall control group (10%, adjusted-HR 2.45 (95%CI, 1.61-3.74), p < 0.001) or the respiratory control group (10%, adjusted-HR 2.93 (95%CI, 1.66-5.17), p < 0.001). Blood oxygen saturation, age, high-sensitivity troponin, c-reactive protein, and lactate dehydrogenase were identified as the strongest predictors of poor outcome available at time of ED presentation in COVID-19 with highly comparable prognostic utility in overall and respiratory controls. In conclusion, patients presenting to the ED with COVID-19 have a worse outcome than controls, even after adjustment for differences in baseline characteristics. Most predictors of poor outcome in COVID-19 were not restricted to COVID-19, but of comparable prognostic utility in controls and therefore generalizable to unselected patients with suspected COVID-19.

Association of Heart Rate Variability With Silent Brain Infarcts in Patients With Atrial Fibrillation.

Purpose: Silent brain infarcts (SBI) are frequently detected in patients with atrial fibrillation (AF), but it is unknown whether SBI are linked to autonomic dysfunction. We aimed to explore the association of autonomic dysfunction with SBI in AF patients. Methods: 1,358 AF patients without prior stroke or TIA underwent brain MRI and 5-min resting ECG. We divided our cohort into AF patients who presented in sinus rhythm (SR-group, n = 816) or AF (AF-group, n = 542). HRV triangular index (HRVI), standard deviation of normal-to-normal intervals, mean heart rate, root mean square root of successive differences of normal-to-normal intervals, 5-min total power and power in the low frequency, high frequency and very low frequency range were calculated. Primary outcome was presence of SBI in the SR group, defined as large non-cortical or cortical infarcts. Secondary outcomes were SBI volumes and topography. Results: Mean age was 72 ± 9 years, 27% were female. SBI were detected in 10.5% of the SR group and in 19.9% of the AF group (p < 0.001). HRVI <15 was the only HRV parameter associated with the presence of SBI after adjustment for clinical covariates in the SR group [odds ratio (OR) 1.67; 95% confidence interval (CI): 1.03-2.70; p = 0.037]. HRVI <15 was associated with larger brain infarct volumes [ß (95% CI) -0.47 (-0.84; -0.09), p = 0.016] in the SR group and was more frequently observed in patients with right- than left-hemispheric SBI (p = 0.017). Conclusion: Impaired HRVI is associated with SBI in AF patients. AF patients with autonomic dysfunction might undergo systematic brain MRI screening to initiate intensified medical treatment. Clinical Trials Gov Identifier: NCT02105844.

Blood Pressure and Brain Lesions in Patients With Atrial Fibrillation.

The association of blood pressure (BP) and hypertension with the presence of different types of brain lesions in patients with atrial fibrillation is unclear. BP values were obtained in a multicenter cohort of patients with atrial fibrillation. Systolic and diastolic BP was categorized in predefined groups. All patients underwent brain magnetic resonance imaging and neurocognitive testing. Brain lesions were classified as large noncortical or cortical infarcts, small noncortical infarcts, microbleeds, or white matter lesions. White matter lesions were graded according to the Fazekas scale. Overall, 1738 patients with atrial fibrillation were enrolled in this cross-sectional analysis (mean age, 73 years, 73% males). Mean BP was 135/79 mm Hg, and 67% of participants were taking BP-lowering treatment. White matter lesions Fazekas ≥2 were found in 54%, large noncortical or cortical infarcts in 22%, small noncortical infarcts in 21%, and microbleeds in 22% of patients, respectively. Compared with patients with systolic BP <120 mm Hg, the adjusted odds ratios (95% CI) for Fazekas≥2 was 1.25 (0.94-1.66), 1.41 (1.03-1.93), and 2.54 (1.65-3.95) among patients with systolic BP of 120 to 140, 140 to 160, and ≥160 mm Hg (P for linear trend<0.001). Per 5 mm Hg increase in systolic and diastolic BP, the adjusted ß-coefficient (95% CI) for log-transformed white matter lesions was 0.04 (0.02-0.05), P<0.001 and 0.04 (0.01-0.06), P=0.004. Systolic BP was associated with small noncortical infarcts (odds ratios [95% CI] per 5 mm Hg 1.05 [1.01-1.08], P=0.006), microbleeds were associated with hypertension, but large noncortical or cortical infarcts were not associated with BP or hypertension. After multivariable adjustment, BP and hypertension were not associated with neurocognitive function. Among patients with atrial fibrillation, BP is strongly associated with the presence and extent of white matter lesions, but there is no association with large noncortical or cortical infarcts. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02105844.

Heart Rate Variability Triangular Index as a Predictor of Cardiovascular Mortality in Patients With Atrial Fibrillation.

Background Impaired heart rate variability (HRV) is associated with increased mortality in sinus rhythm. However, HRV has not been systematically assessed in patients with atrial fibrillation (AF). We hypothesized that parameters of HRV may be predictive of cardiovascular death in patients with AF. Methods and Results From the multicenter prospective Swiss-AF (Swiss Atrial Fibrillation) Cohort Study, we enrolled 1922 patients who were in sinus rhythm or AF. Resting ECG recordings of 5-minute duration were obtained at baseline. Standard parameters of HRV (HRV triangular index, SD of the normal-to-normal intervals, square root of the mean squared differences of successive normal-to-normal intervals and mean heart rate) were calculated. During follow-up, an end point committee adjudicated each cause of death. During a mean follow-up time of 2.6±1.0 years, 143 (7.4%) patients died; 92 deaths were attributable to cardiovascular reasons. In a Cox regression model including multiple covariates (age, sex, body mass index, smoking status, history of diabetes mellitus, history of hypertension, history of stroke/transient ischemic attack, history of myocardial infarction, antiarrhythmic drugs including ß blockers, oral anticoagulation), a decreased HRV index ≤ median (14.29), but not other HRV parameters, was associated with an increase in the risk of cardiovascular death (hazard ratio, 1.7; 95% CI, 1.1-2.6; P=0.01) and all-cause death (hazard ratio, 1.42; 95% CI, 1.02-1.98; P=0.04). Conclusions The HRV index measured in a single 5-minute ECG recording in a cohort of patients with AF is an independent predictor of cardiovascular mortality. HRV analysis in patients with AF might be a valuable tool for further risk stratification to guide patient management. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02105844.

Delayed Diagnosis in Pulmonary Embolism: Frequency, Patient Characteristics, and Outcome.

BACKGROUND: The incidence and the outcomes of pulmonary embolism (PE) missed during emergency department (ED) workup are largely unknown. OBJECTIVES: To describe the frequency, demographics, and outcomes of patients with delayed diagnosis of PE. METHODS: We retrospectively compared patients diagnosed with PE during ED workup (early diagnosis) with patients diagnosed with PE thereafter (delayed diagnosis). Electronic health records (EHR) of 123,560 consecutive patients who attended a tertiary hospital ED were screened. Data were matched with radiology and pathology results from the EHR. RESULTS: Of 1,119 patients presenting to the ED with early workup for PE, PE was diagnosed in 182 patients (80.5%) as early diagnosis. Delayed diagnosis was established in 44 cases (19.5%) using radiology and/or autopsy data. Median age of patients with early diagnosis was significantly lower as compared to delayed diagnosis (67 vs. 77.5 years). Main symptoms were dyspnea (109 patients [59.9%] in early, 20 patients [45.5%] in delayed diagnosis), chest pain (90 patients [49.5%] in early, 8 patients [18.2%] in delayed diagnosis), and nonspecific complaints (16 patients [8.8%] in early, 13 patients [29.5%] in delayed diagnosis). In-hospital mortality was 1.6% in early diagnosis and 43.2% in delayed diagnosis. CONCLUSIONS: Delayed diagnosis of PE carries a worse prognosis than early diagnosis. This discrepancy may arise from either delayed therapy, confounding variables (e.g., older age), or both. Possible reasons for delayed diagnoses are nonspecific presentations and symptoms overlapping with preexisting conditions.

Platelet-Derived Ectosomes Reduce NK Cell Function.

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-ß1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti-TGF-ß1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-ß1-mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti-TGF-ß1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-ß1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell-derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-ß1-dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.

Ectosomes of polymorphonuclear neutrophils activate multiple signaling pathways in macrophages.

Ectosomes are vesicles shed directly from the cell surface. Human polymorphonuclear neutrophils release ectosomes (PMN-Ect) upon their activation. PMN-Ect expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, and down-modulate the inflammatory response of human macrophages and dendritic cells exposed to TLR-2 and -4 ligands. This down-modulation is mediated by PS via the engagement and activation of the Mer receptor tyrosine kinase (MerTK). In the present study, we demonstrate that exposure of macrophages to PMN-Ect activates directly 2 additional pathways, an immediate Ca(2+) flux and a rapid release of TGF-ß1. As expected, the Ca(2+) flux was necessary for the activation of TLR-2 pathway with the release of cytokines. However, MerTK blockade with antibodies did not modify the Ca(2+) flux, indicating an independent activation of Ca(2+) by PMN-Ect. Striking was that the rapid release of TGF-ß1 was independent of the MerTK pathway and did not require a Ca(2+) flux. TGF-ß1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-ß1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-ß1 by macrophages was seen only for PMN-Ect and not for PS-liposomes or erythrocyte ectosomes, which express PS. However, blocking the PS of PMN-Ect inhibited TGF-ß1 release, suggesting that PS expression was necessary although not sufficient for this release. Interestingly, the effects of PMN-Ect pre-exposure were lasting for 24h with the macrophages being less receptive to TLR-2 activation and TGF-ß1 stores remaining low. In sum, PMN-Ect induce several signaling pathways in resting and stimulated macrophages, which include independently the MerTK pathway, Ca(2+) flux and the release of stored TGF-ß1, and each might influence the immunomodulatory effects of macrophages.

Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells.

Microparticles (MP) shed by platelets (PLT) during storage have procoagulant activities, but little is known about their properties to modify inflammation or immunity. In this study, we studied the capacity of MP present in PLT concentrates to alter the function of macrophages and dendritic cells (DC). The size of the purified MP was between 100 and 1000 nm, and they expressed phosphatidylserine; surface proteins of PLT (CD61, CD36, CD47), including complement inhibitors (CD55, CD59), but not CD63; and proteins acquired from plasma (C1q, C3 fragments, factor H). These characteristics suggest that the MP shed by PLT are formed by budding from the cell surface, corresponding to ectosomes. The purified PLT ectosomes (PLT-Ect) reduced the release of TNF-α and IL-10 by macrophages activated with LPS or zymosan A. In addition, PLT-Ect induced the immediate release of TGF-ß from macrophages, a release that was not modified by LPS or zymosan A. Macrophages had a reduced TNF-α release even 24 h after their exposure to PLT-Ect, suggesting that PLT-Ect induced a modification of the differentiation of macrophages. Similarly, the conventional 6-d differentiation of monocytes to immature DC by IL-4 and GM-CSF was modified by the presence of PLT-Ect during the first 2 d. Immature DC expressed less HLA-DP DQ DR and CD80 and lost part of their phagocytic activity, and their LPS-induced maturation was downmodulated when exposed to PLT-Ect. These data indicate that PLT-Ect shed by stored PLT have intrinsic properties that modify macrophage and DC differentiation toward less reactive states.

Ectosomes as immunomodulators.

Considerable progress has been made in recognizing microvesicles as important mediators of intercellular communication rather than irrelevant cell debris. Microvesicles released by budding directly from the cell membrane surface (i.e., ectocytosis) either spontaneously or in response to various stimuli are called shed vesicles or ectosomes. Ectosomes are rightside-out vesicles with cytosolic content, and they expose phosphatidylserine in the outer leaflet of their membrane. Depending on their cellular origin, ectosomes have been associated with a broad spectrum of biological activities. In the light of recent findings, we now know that ectosomes derived from polymorphonuclear leukocytes, erythrocytes, platelets, and tumor cells have profound effects on the innate immune system, as well as on the induction of the adaptive immunity, globally reprogramming cells such as macrophages or dendritic cells toward an immunosuppressive and possibly tolerogenic phenotype. Although the effects observed in the circulation are mainly procoagulant and pro-inflammatory, ectosomes might be anti-inflammatory/immunosuppressive in local inflammation.

Ectosomes released by polymorphonuclear neutrophils induce a MerTK-dependent anti-inflammatory pathway in macrophages.

At the earliest stage of activation, human polymorphonuclear neutrophils release vesicles derived directly from the cell surface. These vesicles, called ectosomes (PMN-Ect), expose phosphatidylserine in the outer membrane leaflet. They inhibit the inflammatory response of human monocyte-derived macrophages and dendritic cells to zymosan A (ZymA) and LPS and induce TGF-ß1 release, suggesting a reprogramming toward a tolerogenic phenotype. The receptors and signaling pathways involved have not yet been defined. Here, we demonstrate that PMN-Ect interfered with ZymA activation of macrophages via inhibition of NFκB p65 phosphorylation and NFκB translocation. The MerTK (Mer receptor tyrosine kinase) and PI3K/Akt pathways played a key role in this immunomodulatory effect as shown using specific MerTK-blocking antibodies and PI3K inhibitors LY294002 and wortmannin. As a result, PMN-Ect reduced the transcription of many proinflammatory genes in ZymA-activated macrophages. In sum, PMN-Ect interacted with the macrophages by activation of the MerTK pathway responsible for down-modulation of the proinflammatory signals generated by ZymA.

Erythrocyte-derived ectosomes have immunosuppressive properties.

Several clinical studies have suggested that blood transfusions are immunosuppressive. Whereas there have been reports describing immunosuppression induced by leukocytes or fragments thereof, the possibility that microparticles, released by erythrocytes during storage, are also involved was not investigated. We present evidence here that such microparticles have all the properties of ectosomes including size, the presence of a lipid membrane, and the specific sorting of proteins. These erythrocyte-derived ectosomes (E-ecto) fixed C1q, which was followed by activation of the classical pathway of complement with binding of C3 fragments. Similarly to ectosomes released by PMN, they express phosphatidylserine on their surface membrane, suggesting that they may react with and down-regulate cells of the immune system. In vitro, they were taken up by macrophages, and they significantly inhibited the activation of these macrophages by zymosan A and LPS, as shown by a significant drop in TNF-alpha and IL-8 release (respectively, 80% and 76% inhibitions). In addition, the effect of E-ecto was not transient but lasted for at least 24 h. In sum, E-ecto may interfere with the innate immune system/inflammatory reaction. Therefore, E-ecto transfused with erythrocytes may account for some of the immunosuppressive properties attributed to blood transfusions.

Polymorphonuclear neutrophil-derived ectosomes interfere with the maturation of monocyte-derived dendritic cells.

Polymorphonuclear neutrophils (PMNs) are a key component of the innate immune system. Their activation leads to the release of potent antimicrobial agents through degranulation. Simultaneously, PMNs release cell surface-derived microvesicles, so-called ectosomes (PMN-Ect). PMN-Ect are rightside-out vesicles with a diameter of 50-200 nm. They expose phosphatidylserine in the outer leaflet of their membrane and down-modulate monocyte/macrophage-activation in vitro. In this study, we analyzed the effects of PMN-Ect on maturation of human monocyte-derived dendritic cells (MoDCs). Intriguingly, exposing immature MoDCs to PMN-Ect modified their morphology, reduced their phagocytic activity, and increased the release of TGF-beta1. When immature MoDCs were incubated with PMN-Ect and stimulated with the TLR4 ligand LPS, the maturation process was partially inhibited as evidenced by reduced expression of cell surface markers (CD40, CD80, CD83, CD86, and HLA-DP DQ DR), inhibition of cytokine-release (IL-8, IL-10, IL-12, and TNF-alpha), and a reduced capacity to induce T cell proliferation. Together these data provide evidence that PMN-Ect have the ability to modify MoDC maturation and function. PMN-Ect may thus represent an as yet unidentified host-factor influencing MoDC maturation at the site of injury, thereby possibly impacting on downstream MoDC-dependent immunity.

Human CD8+ T cells store CXCR1 in a distinct intracellular compartment and up-regulate it rapidly to the cell surface upon activation.

Activation and subsequent differentiation of naive CD8+ T cells lead to the development of memory subsets with distinct homing and effector capacities. On nonlymphoid homing subsets, expression of "inflammatory" chemokine receptors (such as CXCR3, CCR5, CX3CR1, and CXCR1) is believed to promote migration into sites of infection/inflammation. Here we show that CXCR1 can be up-regulated to the cell surface within minutes of activating human CD8+ T cells. No concurrent up-regulation of other inflammatory chemokine receptors was observed. Up-regulation of CXCR1 preferentially occurred on central memory CD8+ T cells-that is, cells with a lymph node homing phenotype-and was functionally relevant. Immunofluorescence microscopy showed CXCR1 to be present in intracellular vesicles that do not significantly colocalize with perforin, RANTES (regulated upon activation normal T cell expressed and secreted), or the lysosomal marker CD63. By contrast, partial colocalization with the Golgi marker GM130, the constitutive secretory pathway marker beta2-microglobulin, and the early endosome marker EEA1 was observed. Up-regulation of CXCR1 did not occur after T-cell receptor cross-linking. By contrast, supernatants from activated neutrophils, but not from monocytes or dendritic cells, induced its up-regulation. These results suggest that CD8+ T cells can rapidly adapt their homing properties by mobilizing CXCR1 from a distinct intracellular compartment.